1. Field of the Invention
This invention relates to an ice seeding apparatus for cryopreservation systems for biological samples such as cells, harvested tissue and cellular biological constructs such as cultured tissue equivalents. Ice seeding in a cryopreservation protocol initiates the formation of ice that is controllable to allow for maximal viability of the tissue or tissue equivalent to be cryopreserved after it has been subsequently thawed. By use of the cryopreservation technology, either cryopreserved harvested tissue or cryopreserved cultured tissue may be stored for indefinite periods of time prior to use. The cultured tissue is an in vitro model of the equivalent human tissue, which, when retrieved from storage, can be used for transplantation or implantation, in vivo, or for screening compounds in vitro.
2. Brief Description of the Background of the Invention
Heretofore, the cryopreservation of cadaver tissue and cultured tissue equivalents for the purposes of preserving the viability of the cells in the tissue has been achieved, but with limited success. Currently, the storage time of cellular biological materials is extended by cooling to xe2x80x9ccryogenicxe2x80x9d temperatures.
The transition from the liquid into the solid state by lowering the temperature of the system can take place either as crystallization (ice), involving an orderly arrangement of water moleciles,. or as vitrification or amorphization (glass formation), in the absence of such an orderly arrangement of crystalline phase. The challenge for a cryobiologist is to bring cells to cryogenic temperatures and then return them to physiological conditions without injuring them.
There are two basic approaches to cryopreservation of cells and tissues: freeze-thaw and vitrification. In freeze-thaw techniques, the extracellular solution is frozen (i.e., in crystalline form), but steps are taken to minimize the intracellular ice formation. In vitrification procedures, there is an attempt to prevent crystalline ice formation throughout the cells and tissue. The former approach is problematic in that if ice crystals are formed inside the cells, they are detrimental to cell viability upon thawing. However, cells could survive a freeze-thaw cycle if they are cooled at controlled rates in the presence of non-toxic levels of cryoprotectants. The latter approach of vitrification seeks to avoid potentially damaging affects of intra- and extracellular ice by depressing ice formation by the addition of very high concentrations of solutes and/or polymers. However, cell damage may occur from long exposure to toxic levels of these additives required for vitrification.
Cryoprotectants protect living cells from the stresses involved in the freezing process. One way cryoprotectants protect cells is by diluting the salt that becomes increasingly concentrated in the unfrozen solution as water is transformed to ice. The amount of ice is dictated by the temperature and initial composition of the solution; whereas the amount of unfrozen fraction is a function of temperature only. Cryoprotectants have several other functions. An important one is that they usually reduce the intracellular ice formation during freezing and thawing of a biological sample. Another function is that they stabilize membranes and proteins. Once the extracellular ice is seeded and the sample is surrounded by the ice phase, it is necessary to cool the sample to a cryopreserved state. The. cooling step is one of the most critical steps in a freeze-thaw protocol. Due to the formation of ice, that is, pure water, the partially frozen extracellular solution is more concentrated than the intracellular compartment. As a consequence, the cell will dehydrate by losing water in an attempt to restore thermodynamic equilibrium. As the system cools, more extracellular ice is generated and the concentration of solutes rises and forces the cells to dehydrate further. There are three characteristics of the cells that control their rate of dehydration One is the cell membrane water permeability; the lower the water permeability, the longer it takes for the cells to dehydrate. Another is the temperature dependence of the cell membrane water permeability; water permeability decreases with decreasing temperatures. The final is cell size; larger cells take longer to -dehydrate than smaller cells. Given that each cell type may have drastically different characteristics, the optimal cryopreservation conditions can vary by orders of magnitude for different cell types.
All solutions will supercool below their freezing point until they find a random nucleation site for crystal formation. When cryopreserving by a freeze-thaw method, ice formation in the extracellular medium should be deliberately initiated by seeding at low degrees of supercooling. If ice formation is not induced by seeding, ice will form spontaneously when the solution is cooled sufficiently far below its equilibrium freezing point. Because this process is random in nature, ice formation will occur at random, unpredictable temperatures. Consequently, survival rates will be highly variable between repeated trials with the same freezing protocol. Furthermore, the extremely rapid crystallization which results when ice forms in a highly supercooled solution can cause damage to cells and tissues. Moreover, it has been shown that if extracellular ice formation is initiated at high degrees of supercooling, the probability of intracellular ice formation is drastically increased. This phenomenon results from the delayed onset of freeze-induced cell dehydration, which results in increased retention of intracellular water, and thus higher likelihood of ice formation in the cell.
Although the exact mechanisms of cell damage during cryopreservation have not yet been completely elucidated, cell survival as a function of cooling rate appear to be qualitatively similar for all cell types and displays an inverted U-shaped curve. Cell survival is low at very slow and very fast cooling rates, and there is an intermediate cooling rate yielding optimal survival. Even though the optimal cooling rate and the width of the curve can vary drastically for different cell types, the qualitative behavior appears to be universal. Faster cooling rates do not allow cells enough time to dehydrate and cells therefore form ice internally. Cell injury at fast cooling rates is attributed to intracellular ice formation. At slow rates of cooling, cell injury is thought to be due to the effects of exposure to highly concentrated intra- and extracellular salt and cryoprotectant solutions or to the mechanical interactions between cells and the extracellular ice.
It is necessary to dehydrate the cells as much as possible before they cross the intracellular ice nucleation curve. It is at this point that water remaining in the cell will nucleate and form ice. The temperature where this will happen is approximately xe2x88x9240xc2x0 C. to xe2x88x9250xc2x0 C. when the cells are slowly frozen in the presence of 1 M to 2 M concentrations of cryoprotectants. It is important to note that the amount of water that to ice inside a cell at this point may be innocuous when frozen, but if not thawed fast enough, rearrangement of ice may kill the cell upon thawing. (The Biophysics of Organ Cryopreservation, Pg. 117-140, edited by David E. Pegg and Armand M. Karow, Jr. NATO ASI Series A: Life Sciences Vol. 147 1987 Plenum Press, New York 233 Spring St., New York, N.Y. 10013).
Other cryopreservation systems, particularly those relating to the freezing of biological samples comprising cells either seed ice by another means, as in chamber spike methods or by use of electronic or mechanical means, or do not seed ice at all.
To seed ice using chamber spike methods, a chamber containing biological samples, such as vials of cells, is quickly cooled to a temperature well below the freezing (i.e., liquid-solid equilibrium temperature) point of the cryopreservation medium then the temperature is raised quickly to near the equilibrium temperature. A drawback to this method is that chamber temperature variations create problems for uniform ice seeding. In many cases, overseeding of ice in the samples occurs resulting in cell damage.
Electronic methods use thermoelectric elements based on semiconductor thermocouples that can controllably produce local cooling. Interfacing these thermoelectric elements with the surface of a container or package containing a biological sample is difficult and variations in effective cooling of the container surface can occur.
Mechanical means of providing localized cooling by use of cold probe, bars or pins to contact the side of a container is problematic in that the process is labor intensive and requires opening the thermal chamber or development of sophisticated mechanisms for guiding the told probe.
Accordingly, it has long been desired to provide a better method of cryopreserving harvested tissue and cultured tissue equivalents to improve cell viability after the tissue has subsequently been thawed. The inventors of the present invention have developed a novel apparatus and method of inducing ice formation in cryopreservation solution, contained in a package with tissue to be frozen, that allows for consistent and reliable seeding of ice of a sufficient amount. The apparatus is standardized and is expandable with additional fixtures added to the apparatus.
The apparatus and method of the present invention provide for cryopreservation of biological specimens such as cells, tissues and tissue equivalents, and maintains their viability after subsequent thawing. Cryopreservation is performed in a freezing chamber at a controlled freezing rate. Tissues and tissue equivalents are perfused with a cryoprotective medium while agitated. Specimens are sealed in a package containing cryoprotective medium and cooled to or slightly below the liquid-solid equilibrium temperature of the medium. At that temperature, the ice seeding is performed, resulting in a seed of ice in the medium. The temperature is held constant for a sufficient amount of time to allow equilibration between the liquid and solid phases. The temperature of the chamber is then lowered at a slow rate to an intermediate temperature; then rapidly to a cryogenic temperature.
In the preferred embodiment, ice seeding is accomplished by discharging a liquefied or chilled gas, preferably freon, from the sprayrails to adjacent racks containing tissue equivalent samples packaged in cryoprotectant. The freon discharged contacts the exterior surface of the package and evaporates. The heat transfer from the package due to the evaporation of the freon results in local cooling in the cryoprotective medium at the freon contact site within the package. Sufficient cooling of the medium at that site causes a degree of ice formation, an ice seed, in the medium. The apparatus for cooling preferably has a vertically-oriented sprayrail with nozzles at a position to be dose to containers when mounted on a removable rack. The system can have multiple sprayrails and valves for directing the fluid to the desired sprayrail.
An advantage of the ice seeding system is the ability to consistently form a seed of ice in a plurality of sealed packages containing cryoprotective medium and tissue or equivalents thereof. The cryopreservation apparatus and method of the present invention can be used in the manufacturing process for storing and shipping of these tissues while frozen. The tissues are rendered viable when thawed.
The use of the ice seeding system in the cryopreservation process has demonstrated an application in the manufacturing process of living tissue equivalents. Prior to this invention, harvested tissue and living tissue equivalents had limited shelf-life and, subsequently, their window of use is short, resulting in much waste. There is a need to preserve such tissues for extended periods of time, as in shipping and storage, until their use. Previous attempts to freeze or freeze dry these tissues have been met with limited success and have compromised their use for grafting, in vivo, or for in vitro testing. The ability to use these tissues in a viable state represents an exceptional advantage of the present invention.